Ionizing radiation (IR) is significantly toxic to cells as well as being a potent mutagen and carcinogen. Humans and other mammals display considerable population variation in their response to the lethal effects of ionizing radiation. Historically, there has been much interest in elucidating the genetic bases for this variation both because of the insights to be gained into the cellular pathways controlling DNA damage responses and because of the potential to utilize this information to personalize radiation therapy to the specific sensitivities of individual patients. For more than 20 years, we have studied the genetics of radiosensitivity in humans, focusing on recessive genetic disorders that result in the most extreme IR hypersensitivity phenotypes. These studies have included the localization and fine mapping of ATM, the gene mutated in A-T, the mapping and cloning of the NBN gene mutated in NBS, and the description of Ligase IV Syndrome, in which patients display clinical features similar to NBS but have biallelic inactivating mutations in the LIG4 gene. During the course of these studies, we have accumulated cell lines from patients referred to us for diagnostic testing for A-T or NBS in whom no mutations in either the ATM or NBN genes could be found after exhaustive screening. These cell lines display significant radiosensitivity in a standard colony survival assay comparable to that of A-T cell lines. These radiosensitive lymphoblastoid cell lines (RS-LCL) constitute a novel resource for identifying and understanding the mechanisms of human cellular radiation hypersensitivity. We hypothesize that individual RS-LCLs contain monogenic mutations in different genes whose contributions to cellular DNA damage responses are known, or may as yet be unrecognized. We further postulate that, by analogy with known disorders such as A-T or NBS, the RS-LCLs derive from individuals with as yet undescribed recessive genetic disorders caused by deleterious biallelic mutations in single genes. Our previous attempts to identify mutations among these cell lines by candidate gene approaches have yielded results that are consistent with this model; we identified two siblings with biallelic LIG4 mutations and a second, as yet unreported, patient with biallelic mutations in RNF168. These results suggest that the RS panel is enriched for patients with inherited defects in DNA damage responses but that the causes of radiosensitivity among cell lines in the panel are diverse; candidate gene studies would be an inefficient approach to identify the genetic lesions underlying the radiosensitivity in the majority of these cell lines. Fortunately, it is now possible to identify mutations in monogenic disorders represented by one or a few affected individuals through newly developed exome sequencing approaches. We propose here to apply exome sequencing to identify the causative mutations in our panel of RS-LCLs in a staged approach; sequencing a representative sampling of the RS-LCLs, seeking confirmation by screening the identified genes in the remainder of the panel and finally, complementing the radiosensitivity of individual cell lines by transfecting wild type copies of the mutated genes.